重组人αB-晶体蛋白对大鼠视神经损伤修复作用的研究
发布时间：2018-07-21 10:30
【摘要】：视神经损伤是一种视功能损伤严重,预后极差的眼外伤类型和致盲原因。因为中枢神经系统组织损伤后不能再生,和缺乏修复功能,目前尚无确切的治疗方式。我们的研究小组近十年的研究结果表明晶体损伤后释放的晶体蛋白对视神经损伤有修复和再生作用,这一现象为视神经损伤的治疗带来了希望。近年的国内外研究表明晶体蛋白中的αB-晶体蛋白是这一家族中最活跃的小分子蛋白,属于热休克蛋白家族,不仅可以对变性神经组织有保护和促进修复作用,还具有抑制炎症、应激反应,维护细胞骨架蛋白和骨架网络稳定性,及抵抗缺氧、高温、反射线及药物等因素诱导的细胞凋亡的作用。但由于αB-晶体蛋白不能从提取的天然α-晶体蛋白中分离出来,重组人αB-晶状体蛋白是开发应用的唯一途径。国外文献报道重组αB-晶体蛋白用于治疗多发性硬化动物实验取得了良好的疗效。而重组人αB-晶体蛋白全身应用,是否具有视神经保护作用未见文献报道。由此,探讨如何重组有高活性的人αB-晶体蛋白,明确重组人αB-晶体蛋白静脉应用,对视神经保护作用,并对全身重要器官和行为学是否有影响,可能为重组人aB-晶体蛋白临床应用奠定基础,为临床治疗视神经损伤提供新的途径。 【目的】 建立重组蛋白表达体系,重组人αB-晶体蛋白和有更强分子伴侣活性的Trx-αB-crystallin融合蛋白;通过建立大鼠视神经钳夹伤模型,进一步明确重组人αB-晶体蛋白对视神经损伤后视网膜神经节细胞的保护作用;在此基础上,通过动物模型电生理检查分析重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白治疗视神经损伤的疗效,并通过显微组织学检查明确静脉应用重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白对全身重要脏器的影响,为开发重组人αB-晶体蛋白药物治疗视网膜神经节细胞损伤和视神经损伤奠定实验基础。 【方法】 1、根据已知人αB-晶体蛋白基因序列构建目的基因,并建立目的基因载体质粒pET32a和pET28a,通过BL21大肠杆菌细胞为宿主的表达系统,IPTG诱导表达后纯化,得到高纯度的重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白,通过免疫印迹和蛋白质谱分析鉴定重组蛋白。并利用小分子热休克蛋白胰岛素活性实验,验证重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白的体外活性,同时,通过对比研究观察两者分子伴侣活性强弱。 2、以成年Long Evans大鼠为研究对象,建立视神经钳夹损伤模型,静脉注射重组人αB-晶体蛋白、Trx-αB-crystallin融合蛋白、Trx蛋白和生理盐水。通过大脑上丘和外侧膝状体荧光金逆行标记和视网膜铺片的免疫组化染色方法,分别于损伤后2周和4周观察视网膜内存留有活性的视网膜神经节细胞,并定量分析和比较各组视网膜神经节细胞数量。 3、以成年Long Evans大鼠建立视神经钳夹损伤模型,静脉注射重组人αB-晶体蛋白、Trx-αB-crystallin融合蛋白、Trx蛋白和生理盐水。利用闪光视觉诱发电位检测方法,于损伤后2、4周观察各组大鼠视功能情况,探讨重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白对视神经损伤后视功能修复的影响。 4、利用行为学方法,观察上述各组实验动物于损伤后2、4周的行为学变化,并在4周时处死大鼠,取心、肝、脾、肾重要脏器行组织病理学检查,评估静脉应用重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白的安全性。 【结果】 1、构建的载体质粒pET32a-αB-crystallin和pET28a-αB-crystallin,通过BL21大肠杆菌细胞表达和纯化可以得到高纯度的重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白。 2、重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白均具有分子伴侣活性,且Trx-αB-crystallin融合蛋白的活性大于重组人αB-晶体蛋白。 3、视神经钳夹伤后荧光金逆行标记示,静脉注射重组人αB-晶体蛋白组和Trx-αB-crystallin融合蛋白组在损伤后2周和4周与对照组相比均有较多的具有轴浆运输功能的视网膜神经节细胞存活,统计学差异显著(P0.05)。 4、视网膜神经节细胞免疫组化荧光染色示,视神经钳夹伤后2周和4周静脉注射重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白组较Trx蛋白和生理盐水组有更多的视网膜神经节细胞存活,统计学差异显著(P0.05)。 5、视神经钳夹伤后2周脉注射重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白组大鼠较Trx蛋白和生理盐水组视功能检查(闪光视觉诱发电位)示P1波波幅较大,差异显著(P0.05)。但4周时,各组间P1波波幅和潜时差异不显著。 6、大鼠视神经钳夹伤后4周,重组人αB-晶体蛋白、Trx-αB-crystallin融合蛋白组大鼠、Trx蛋白和生理盐水组行为学检测无显著性差异,且各组重要脏器组织学检查未见明显异常。 【结论】 1、大肠杆菌表达体系可以成功表达有分子伴侣活性的重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白。 2、重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白在视神经损伤后修复中发挥重要作用。通过静脉多次给药,可明显减少视神经损伤引起的视网膜节细胞死亡,保存更多有轴浆运输功能的视网膜神经节细胞,提示重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白有促进视神经损伤修复的作用,并可以在损伤早期有效保护视功能。 3、全身静脉应用重组人αB-晶体蛋白和Trx-αB-crystallin融合蛋白安全,不会引起重要脏器病变和行为学改变。 4、Trx-αB-crystallin融合蛋白中Trx标签可以增强αB-晶体蛋白的分子伴侣活性,但不能提高αB-晶体蛋白保护功能,提示αB-晶体蛋白的分子伴侣活性和保护细胞功能可能不完全是一个功能结构域。
[Abstract]:In recent years , it has been shown that 伪 - B - crystallin is the most active small molecule protein in the family of optic nerve .
Purpose of the project
The expression system of recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein with stronger molecular partner activity were established . The effect of recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein on optic nerve injury was further clarified by establishing the rat optic nerve clamp injury model . The effects of recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein on the whole body organ were analyzed by electrophysiological examination of animal model .
Methodology
1 . The recombinant human 伪B - crystallin and Trx - 伪B - allallin fusion protein were induced by IPTG . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein were purified by IPTG . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein were identified by Western blot and protein analysis .
2 . Using adult Long Evans rats as the research object , the optic nerve clamp injury model was established , the recombinant human 伪B - crystallin , Trx - 伪B - allallin fusion protein , Trx protein and physiological saline were injected intravenously . The retinal ganglion cells were observed in the retinal memory at 2 and 4 weeks after injury , and the number of retinal ganglion cells in each group was analyzed and compared .
3 . A model of optic nerve clamp injury was established in adult Long Evans rats . The recombinant human 伪B - crystallin , Trx - 伪B - allallin fusion protein , Trx protein and physiological saline were injected intravenously . The effects of recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein on the restoration of optic nerve injury were investigated in 2 and 4 weeks after injury .
4 . Using the behavioral method , the behavioral changes of 2 and 4 weeks after injury were observed and the safety of the recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein was assessed by histopathological examination of the rat , heart , liver , spleen and kidney in 4 weeks .
The result is not valid .
1 . The recombinant human 伪B - crystallin and Trx - 伪B - allallin fusion protein can be obtained by the expression and purification of E . coli cells .
2 . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein all have molecular partner activity , and the activity of Trx - 伪B - crystallin fusion protein is greater than that of recombinant human 伪B - crystallin .
3 . Compared with the control group , the recombinant human 伪B - crystallin group and Trx - 伪B - crystallin fusion protein group had more retinal ganglion cell survival than the control group at 2 and 4 weeks after injury , and the statistical difference was significant ( P0.05 ) .
4 . Immunohistochemical staining of retinal ganglion cells showed that the recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein group had more retinal ganglion cells survival after 2 and 4 weeks after the optic nerve clamp injury , and the statistical difference was significant ( P0.05 ) .
5 . After optic nerve clamp injury 2 weeks after injury , the recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein group showed a larger amplitude of P1 wave than that of Trx protein and physiological saline group ( P0.05 ) . But at 4 weeks , there was no significant difference between P1 wave amplitude and latent time difference among the groups .
6 . There was no significant difference in the behavior of recombinant human 伪B - crystallin , Trx - 伪B - allallin fusion protein group , Trx protein and physiological saline group at 4 weeks after optic nerve clamp injury in rats , and no significant abnormality was seen in histological examination of major organs in each group .
Conclusion
1 . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein expressed by the expression system of E . coli were successfully expressed .
2 . The recombinant human 伪B - crystallin and Trx - 伪B - allallin fusion protein play an important role in the repair of optic nerve injury . The retinal ganglion cells caused by optic nerve injury can be significantly reduced by intravenous administration of multiple doses , and more retinal ganglion cells with axonal transport function are preserved . It is suggested that the recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein have the effect of promoting the repair of optic nerve injury , and can effectively protect the visual function during early injury .
3 . The safety of recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein in systemic vein will not cause major organ changes and behavioral changes .
4 銆，
本文编号：2135232
[Abstract]:In recent years , it has been shown that 伪 - B - crystallin is the most active small molecule protein in the family of optic nerve .
Purpose of the project
The expression system of recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein with stronger molecular partner activity were established . The effect of recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein on optic nerve injury was further clarified by establishing the rat optic nerve clamp injury model . The effects of recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein on the whole body organ were analyzed by electrophysiological examination of animal model .
Methodology
1 . The recombinant human 伪B - crystallin and Trx - 伪B - allallin fusion protein were induced by IPTG . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein were purified by IPTG . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein were identified by Western blot and protein analysis .
2 . Using adult Long Evans rats as the research object , the optic nerve clamp injury model was established , the recombinant human 伪B - crystallin , Trx - 伪B - allallin fusion protein , Trx protein and physiological saline were injected intravenously . The retinal ganglion cells were observed in the retinal memory at 2 and 4 weeks after injury , and the number of retinal ganglion cells in each group was analyzed and compared .
3 . A model of optic nerve clamp injury was established in adult Long Evans rats . The recombinant human 伪B - crystallin , Trx - 伪B - allallin fusion protein , Trx protein and physiological saline were injected intravenously . The effects of recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein on the restoration of optic nerve injury were investigated in 2 and 4 weeks after injury .
4 . Using the behavioral method , the behavioral changes of 2 and 4 weeks after injury were observed and the safety of the recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein was assessed by histopathological examination of the rat , heart , liver , spleen and kidney in 4 weeks .
The result is not valid .
1 . The recombinant human 伪B - crystallin and Trx - 伪B - allallin fusion protein can be obtained by the expression and purification of E . coli cells .
2 . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein all have molecular partner activity , and the activity of Trx - 伪B - crystallin fusion protein is greater than that of recombinant human 伪B - crystallin .
3 . Compared with the control group , the recombinant human 伪B - crystallin group and Trx - 伪B - crystallin fusion protein group had more retinal ganglion cell survival than the control group at 2 and 4 weeks after injury , and the statistical difference was significant ( P0.05 ) .
4 . Immunohistochemical staining of retinal ganglion cells showed that the recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein group had more retinal ganglion cells survival after 2 and 4 weeks after the optic nerve clamp injury , and the statistical difference was significant ( P0.05 ) .
5 . After optic nerve clamp injury 2 weeks after injury , the recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein group showed a larger amplitude of P1 wave than that of Trx protein and physiological saline group ( P0.05 ) . But at 4 weeks , there was no significant difference between P1 wave amplitude and latent time difference among the groups .
6 . There was no significant difference in the behavior of recombinant human 伪B - crystallin , Trx - 伪B - allallin fusion protein group , Trx protein and physiological saline group at 4 weeks after optic nerve clamp injury in rats , and no significant abnormality was seen in histological examination of major organs in each group .
Conclusion
1 . The recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein expressed by the expression system of E . coli were successfully expressed .
2 . The recombinant human 伪B - crystallin and Trx - 伪B - allallin fusion protein play an important role in the repair of optic nerve injury . The retinal ganglion cells caused by optic nerve injury can be significantly reduced by intravenous administration of multiple doses , and more retinal ganglion cells with axonal transport function are preserved . It is suggested that the recombinant human 伪B - crystallin and Trx - 伪B - crystallin fusion protein have the effect of promoting the repair of optic nerve injury , and can effectively protect the visual function during early injury .
3 . The safety of recombinant human 伪 - B - crystallin and Trx - 伪B - crystallin fusion protein in systemic vein will not cause major organ changes and behavioral changes .
4 銆，
本文编号：2135232
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